In vitro kinase assay

In vitro kinase assay

Used in Yu et al., Elife. 2022 Apr 11;11:e74531.

1. Prepare 20× kinase buffer, aliquot and store at -20 ^oC. 

            20× kinase buffer: 1 M Tris-HCl pH 7.5, 2 mM EDTA and 40 mM DTT 

2. Prepare fresh 10× ATP regeneration system (ARS) for each assay, keep on ice.

            10× ARS: 100 mM MgCl₂, 10 mM ATP, 100 mM creatine phosphate and 2 mg/mL creatine kinase

3*. Prepare 4× kinase mixture, keep on ice

            0.8 µM Hrr25 (wild type or K38A mutant), 0.8 µCi/µL γ-³²P-ATP, 4× ARS and 4× kinase buffer; 

            used at 3 µL per 12 µL reaction in PCR tubes

4. Prepare 4× substrate mixture, keep on ice

            2 µM Atg36 or 4 µM Atg19, or 2 µM Atg36 and Atg19 diluted with 25 mM HEPES pH 7.5, 150 mM NaCl, and 1 mM DTT

            used at 3 µL per 12 µL reaction in PCR tubes

5. Prepare 2× ATPase mixture, keep on ice

            400 nM ATPases (wild-type Pex1/6 and Walker B mutant (E832A), wild-type Cdc48 and D2 mutant (E588Q)

            used at 6 µL per 12 µL reaction in PCR tubes.

6. Mix 4× substrate with 2× ATPase mixture, pre-incubate the substrate/ATPase mixture on a 37 ^oC heating block for 5 min

7*. Pre-incubate the 4× kinase mixture on a 37 ^oC heating block for 5 min

8*. Add substrate/ATPase into kinase mixture to start the reaction

9*. Take 2 µL sample at designed time points (e.g., 0.5, 3, 6, 10 and 15 min), mix the samples immediately with 15 µL 1× SDS-PAGE loading buffer, 95 ^oC denaturation for 5 min

10*. Load the sample (~17 µL) onto SDS-PAGE gels (Novex™ WedgeWell™ 4 to 20%, Tris-Glycine, 1.0 mm, Mini Protein Gels), electrophoresis at 200 V for 55 min

11*. Soak the protein gels in gel fixing solution (50% v/v ethanol and 10% v/v acetic acid in water), shake at room temperature for 15 min.

12*. Soak the protein gels in gel drying solution (45% v/v ethanol and 5% v/v glycerol in water), shake at room temperature for 15 min.

13*. Dry the gels with Model 583 Gel Dryers (Bio-Rad)

14*. Expose the dried gels to phosphorimager cassettes overnight

15. Quantify the ³²P-labeled species of Atg36 or Atg19 with FIJI software.

*. ³²P related operation. wear appropriate personal protective equipment